Sterilization-I

Dr. Gunjan Priyam

Introduction
Microorganisms are ubiquitous in nature.
They are found in the surroundings, or inanimate objects
and on the surface of the human body.
Since , they are responsible for infection , contamination
and decay, it is necessary to remove or destroy them in
healthcare settings and other human habitats.
How can microorganism be killed?
1. Denaturation of proteins(eg. Wet heat, ethylene oxide)
2. Oxidation (eg. Dry heat , hydrogen peroxide)
3. Filtration
4. Interruption of DNA synthesis/repair(eg.radiation)
5. Interference with protein synthesis (eg. Bleach)
6. Disruption of cell membrane( eg. Phenols)
Definitions
Sterilisation: The process by which an article, surface or
medium is freed of all living microorganisms in the
vegetative or spore is called sterilisation.

Disinfection: The process of destruction of all pathogenic

organisms is called disinfection. Some may not be able to
destroy spores.
Other definition:
Asepsis: It is the state of complete absence of viable
pathogenic microorganisms in any environment.
Antiseptics: Agents that can be safely applied on the skin
or mucous membrane to prevent infection by inhibiting the
growth of bacteria are called antiseptics .
Decontamination: It is the process of rendering an article,
area, object or body surface free of contaminants including
microbial, chemical, radioactive and other hazardous
material.
Bactericidal agents or (germicide): These are substances
that can kill bacteria.

Bacteriostatic agents: These are substances that prevent

the multiplication of bacteria but do not necessarily kill
them.
Spaulding’s classification
Methods of sterilization
Physical method
1.Heat
a) Dry method: Red heat
Flaming
Incineration
Hot air oven
Infra red light
a) Red heat: Inoculating loops, wires, points of forceps, searing spatula,
needles are kept in the flame till they are red hot.

b) Flaming: Mouths of bottles , culture tubes , glass slides , cover slips,

scalpels and needles are kept in the flame without making it red hot.

c) Incineration: Those articles which are contaminated and can not be

reused like plastic hypodermic syringes , cotton, surgical dressing
materials etc. are burnt to sterilize them.

d) Infra red light: In this method sterilization is done by heat and not be
radiation .Material to be sterilized are put on mechanically carry belt
going through a tunnel having temp. of 180 degree celcius by infra red.
Holding time is 71/2 mins.
e) Hot air Oven
Most widely used method of sterilisation by dry heat.
Sterilisation is achieved by conduction.
Heat is absorbed by the surface of the item to be sterilised ,
which then penetrates to the centre , until the entire item
reaches the desired temp.
 Moist heat
Destroys cells by causing the following changes:
Moist heat results in the denaturation and coagulation of
proteins.
Advantage of steam is the latent heat that is liberated . When
this latent heat condenses on a cooler surface , it raises the
temp. of that surface , thereby denaturing its components.

In the case of spores , steam condenses on them , increasing

their water content and resulting in the ultimate hydrolysis and
breakdown of bacterial protein.
Sterilisation at temperatures below 100
1. Pasteurisation of milk : Milk is heated at either 63 °C for 30
min. (the holder method) or 72°C for 15-20 seconds ( the flash
process) , followed by rapid cooling to 13 °C or lower.
All these methods, all non-sporing pathogens such as
mycobacteria , brucellae and salmonellae are destroyed.
2. Inspissation: Media such as Lowenstein-Jensen and Loffler’s
serum are rendered sterile by heating them at 80-85 °C for half an
hour on 3 successive days in an inspissater.
3.Vaccine bath: Bacterial vaccines are heat-inactivated at 60 °C for 1hr in
special vaccine baths.
 Serum or body fluids containing coagulable proteins can be
sterilised by heating them for 1hr at 56 °C in a water bath on
several successive days.
Sterilisation at 100 °C
1. Boiling: Vegetative bacteria are killed almost immediately
at 90 °C-100 °C but bacterial spores can withstand long
periods of boiling.
2. Tyndallisation or intermittent sterilisation: This method is
used for media containing sugars or gelatin . The media
is exposed to a temp. of 100 °C for 20 mins. On 3
successive days.
The principle of this method is that the first exposure kills
all vegetative bacteria, while the spores , which germinate
subsequently (during the resting period) .
Sterilisation at >100 °C using steam under
pressure
Autoclave (Steam steriliser) :
•The autoclave works on the principle of moist heat
sterilization where steam under pressure is used to sterilize
the material present inside the chamber.
•The high pressure increases the boiling point of water and
thus helps achieve a higher temperature for sterilization.
an autoclave is run at a temperature of 121°C for at least 30
minutes by using saturated steam under at least 15 psi of
pressure.
 Applications
Autoclave is particularly useful for media-containing water that
cannot be sterilized by dry heat. It is the method of choice for
sterilizing the following:
Surgical instruments
Culture media
Autoclavable plastic containers
Plastic tubes and pipette tips
Solutions and water
Biohazardous waste
Glassware (autoclave resistible)
Types of autoclave
Based on size
Large-scale autoclave: It is more significant in size.
Some may have double chambers and can have 500 liters
to more than 1500 liters chamber. It is suitable for
hospitals and clinical and research laboratories.
Small-scale autoclave: It is smaller in size. It has
chambers that can simultaneously fit 20-300 liters of
autoclavable materials. It is suitable for university and
college laboratories.
 Types of Autoclave on the basis of Working
Principle
Gravity displacement autoclave: The hot steam enters the chamber and
forces all the air through a vent. It is unsuitable for autoclave bags because it
creates air pockets. It is generally of two types; horizontal and vertical
autoclave.
Horizontal autoclave: The door/lid of this type of autoclave open outwards
towards the handler. It is usually available in large sizes.
Vertical autoclave: The autoclavable material is loaded from the top side of the
autoclave. It is usually available in small sizes.
Positive pressure displacement autoclave: Here, the steam is generated in
a separate steam generator unit, and then the moisture is transferred into the
autoclave. It is faster because it takes only a few seconds to generate steam.
Negative pressure (vacuum) displacement autoclave: In this type of
autoclave, a vacuum generator creates a vacuum that removes air inside the
chamber before beginning the sterilization cycle. This type of autoclave has
 Precautions

 Never autoclave any liquid in a sealed container.

The following precautions should be taken while using an autoclave.
 Autoclave should not be used for sterilizing waterproof materials, such as oil
and grease, or dry materials, such as glove powder
 Materials are loaded in, such a way that it allows efficient steam penetration
(do not overfill the chamber). It is more efficient and safer to run two
separate, uncrowded loads than one crowded one.
 Wrapping objects in aluminum foil is not recommended because it may
interfere with steam penetration. Articles should be wrapped in materials that
allow steam penetration.
 Materials should not touch the sides or top of the chamber
 The clean items and the wastes should be autoclaved separately.
 Polyethylene trays should not be used as they may melt and cause damage
to the autoclave.
 Sterilization control

Modern autoclaves have devices to maintain proper

pressure and record internal temperature during operation.
Regardless of the presence of such a device, autoclave
pressure should be checked periodically and maintained.
Several methods are available to ensure that autoclaving
achieves sterility. The effectiveness of the sterilization done
by autoclave can be monitored by:
1) Biological indicator
2) Autoclave tapes
3) Thermocouple
4) Browne’s tube
 Biological indicator

Spores of Geobacillus stearothermophilus (formerly called Bacillus

stearothermophilus) are the best indicator because they are resistant to
steam.
 Their spores are killed in 12 minutes at 121°C.
The Centers for Disease Control (CDC) recommends weekly autoclaving of a
culture containing heat resistant endospores of Geobacillus
stearothermophilus, to check autoclave performance.
 The spore strip and an ampule of medium enclosed in a soft plastic vial are
available commercially.
 The vial is placed in the center of the material to be sterilized and is
autoclaved. Then the inner ampule is broken, releasing the medium, and the
whole container is incubated.
 If no growth appears in the autoclaved culture, sterilization is deemed
effective.
 Autoclave tapes
 Adhesive-backed paper tape with heat-sensitive chemical indicator
marking that changes color or display-diagonal stripes, the words
“sterile” or “autoclaved” when exposed to effective sterilization
temperature (121°C) are used to check the efficacy of autoclaves.

 These tapes are placed inside and near the center of large packages
because heat penetration in those areas ensures proper heat
penetration (For example, when a large piece of meat is roasted, the
surface can be well done while the center may still remain unheated, and
if the center is sufficiently heated then it means the desired temperature
is achieved).

 Autoclave tapes are not fully reliable because they do not indicate how
long appropriate conditions were maintained.
Thermocouple and Browne’s tube.
 Thermocouple is a temperature measuring device that
records the temperature by a potentiometer.
Browne’s tube (invented by Albert Browne in 1930)
contains a heat-sensitive red dye that turns green after
being exposed to a certain temperature for a definite
period of time.
Conversion of dye color gives information about the
duration of time and temperature.
Filtration
Useful for substances which get damaged by heat.
To sterilize sera , sugars and antibiotic solutions.
To obtain bacteria free filtrates of clinical samples.
Purification of water.
This method is commonly used for sensitive
pharmaceuticals and protein solutions in biological
research.
A filter with pore size 0.2µm will effectively remove
bacteria ( for virus 20 nanometer)
Prions are not removed by filtration.
Types of filters
1. Candle filters
2. Asbestos filters
3. Sintered glass filters
4. Membrane filters
5. Air filters eg. HEPA filter used in microbiology biosaftey
cabinet
6. Syringe filters
 Radiation
Two types of radiation:
1. Ionizing radiation : eg Gamma rays , X-rays. (Cold
sterilization)
 Used for sterilising plastics, syringes, swabs , catheters, oils ,
fabric and metal.

1. Non-Ionizing radiation: eg. UV rays , Infrared rays. (HOT

sterilization)
 Infrared: Used for rapid mass sterilisation of pre-packed items
such as syringes , catheters.
 Uv rays : Used for disinfecting enclosed areas such as
Operation theatres , Bio-safety cabinets and laboratory